Fig. 1
Wound repair assay design and characterization of wound repair in Stentor coeruleus. A Parallelized guillotine device, with inlets (a) and (b) used for injecting media and cells, respectively, and outlet (c). To parallelize cell cutting, 8 radially arranged guillotines converged in a well leading to outlet (c). B Schematic diagram of the wound repair assay. (a), (b), and (c) refer to the inlets/outlets. (i–iv) are steps in the assay (see Methods). C Cell fluorescence for unwounded cells (controls) (N = 46 cells) and wounded cells (Regime 1, 4 s post-wounding) (N = 41 cells), using the wound repair assay. Data combined from 3 biological replicates per experimental group, with mean lines shown. Representative brightfield and fluorescence images shown. The dashed line at cell fluorescence = 1200 AU indicates the Ithreshold used to distinguish wounded from healed cells. D ROC (receiver operating characteristic) curve for the wound repair assay. Representative images of cells wounded in E Regime 1 and F Regime 2: illustration of typical wound locations (arrows) in a cut cell fragment (left panel), brightfield (top panels), and corresponding fluorescence images (bottom panels) of wounded cells, fixed and stained with Sytox Green at different time points post-wounding. All fluorescence images were scaled equally (100–10,000 AU). Scale bars in C, E, and F are 200 μm. G Fraction of cells healed, using the wound repair assay. Data shown as mean (stars) of N ≥ 3 biological replicates (dots) and fit to a one-phase exponential function using mean datapoints in addition to the 24-h (86,400 s) survival rate, to aid extrapolation. The control line shows the mean fraction of unwounded control cells below Ithreshold, and the shaded region indicates standard deviation (SD) (3 biological replicates). Total N = 31–65 cells per experimental condition. H Survival rate of cells wounded in Regimes 1 and 2, 24 h after wounding, shown as mean (bar) of 3 biological replicates (dots) (N = 22–34 cells per replicate)